Targeting the mitochondrial lysophosphatidic acid-producing enzyme glycerol-3-phosphate acyltransferase 1 in ovarian cancer using a stable knockdown system and the small molecule inhibitor FSG67

Abstract

Ovarian cancer is one of the most commonly detected cancers in women. The lack of efficient screening methods complicates early disease detection and thus ovarian cancer is often diagnosed at later stages when more severe symptoms due to metastasis are present and the five-year relative survival rate is only 29%. Recently lysophosphatidic acid (LPA) was reported to play an important role in different diseases, including ovarian cancer. So far, mainly the role of extracellular LPA as a signaling lipid has been studied; however, LPA is also produced intracellularly, and as opposed to extracellular LPA, little is known of its role as a signaling lipid. Intracellular LPA can be produced by several metabolic pathways and enzymes, including the glycerol-3-phosphate acyltransferase (GPAT) enzymes. The glycerol-3-phosphate acyltransferase 1, mitochondrial (GPAM) has been linked to several diseases, including metabolic disorders and cancer. For example, GPAM expression was associated with worse outcome in human ovarian cancer patients, and its silencing reduced tumor growth in a subcutaneous xenograft mouse model. However, the contribution of GPAM to ovarian cancer metastasis has not yet been studied in vivo. Thus, in this PhD thesis, the effect of silencing GPAM with a stable knockdown system, appropriate for in vivo studies, as well as pharmacologically inhibiting its enzymatic activity with the small molecule inhibitor FSG67 was evaluated both in vitro and in vivo. Luciferase-expressing ES-2 ovarian cancer cell lines were generated to be used for in vivo imaging of peritoneal metastasis. The most promising luciferase-expressing ovarian cancer cell line was then chosen to generate stable GPAM knockdown cell lines, suitable for in vivo studies. The stable GPAM knockdown was first evaluated in vitro, revealing that silencing GPAM decreased collective cell migration and the number of colonies formed, but had no effect on colony size nor anoikis resistance, and did not strongly influence individual cell migration. In a subsequent experiment using an in vivo model of peritoneal metastasis, stable GPAM knockdown in ES-2 cells slowed tumor growth suggesting a possible role for GPAM in ovarian cancer metastasis. Moreover, the effect of inhibiting GPAM with the small molecule GPAT inhibitor FSG67 was also evaluated, demonstrating that FSG67 altered the formation of colonies and reduced individual cell migration, collective cell migration and resistance to anoikis in ovarian cancer cells. Additionally, toxicology and pharmacokinetic studies of FSG67 in vivo revealed the compound to be suitable for future in vivo studies, such as antitumor efficacy studies of the compound. To gain further insight into the mechanism behind GPAM’s role in the observed cancer cell phenotypes, its enzymatic product, LPA was studied as a possible autocrine and/or paracrine signaling molecule using mass spectrometry-based measurements of labeled LPA, demonstrating that intracellular LPA was not transported into the extracellular space. In addition, intracellular binding partners of LPA were evaluated on the protein level. Therefore, an LPA-protein pull-down assay was established and subsequently used with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) based proteomics, identifying S100 calcium-binding protein A7 (S100A7), BPI fold-containing family B member 1 (BPIFB1), suprabasin (SBSN) and actin (ACTB/ACTG) as intracellular LPA-binding protein candidates. In summary, this work shows that silencing GPAM or inhibiting GPAM with FSG67 hinders cancer-relevant processes and that both approaches are suitable for in vivo studies of ovarian cancer metastasis. Furthermore, it identified intracellular LPA-binding protein candidates to be followed up in future studies.